Optogenetic magnetic resonance imaging

ABSTRACT

Disclosed herein are systems and methods involving the use of magnetic resonance imaging and optogenetic neural stimulation. Aspects of the disclosure include modifying a target neural cell population in a first region of a brain to express light-responsive molecules. Using a light pulse, the light-responsive molecules in the target neural cell population are stimulated. Multiple regions of the brain are scanned via magnetic resonance imaging. The scans allow for observation of a neural reaction in response to the stimulation in at least one of the multiple regions of the brain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.13/847,653, filed Mar. 20, 2013, now U.S. Pat. No. 8,834,546, which is acontinuation of U.S. patent application Ser. No. 13/299,727, filed Nov.18, 2011, now U.S. Pat. No. 8,696,722, which application claims benefitunder 35 U.S.C. §119 to U.S. Provisional Patent Application Ser. No.61/416,143 filed on Nov. 22, 2010; U.S. patent application Ser. No.13/847,653, U.S. patent application Ser. No. 13/299,727, and U.S. patentapplication Ser. No. 61/416,143 and its Appendix, including thereferences cited therein, are hereby incorporated by reference in theirentirety.

FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government Support under Grant No.1K99EB008738-01 awarded by the National Institute of Health. The U.S.Government has certain rights in this invention.

BACKGROUND

Blood oxygenation level-dependent functional magnetic resonance imaging(BOLD fMRI) is a widely used technology for non-invasive whole brainimaging. BOLD signals reflect complex changes in cerebral blood flow(CBF), cerebral blood volume (CBV), and cerebral metabolic rate ofoxygen consumption (CMRO₂) following neuronal activity. However, theneural circuits that trigger BOLD signals are not completely understood,which may confound fMRI interpretation. Candidate circuit elements fortriggering various kinds of BOLD signals include excitatory neurons,mixed neuronal populations, astroglia, and axonal tracts or fibers ofpassage. Understanding the neural circuits that give rise to BOLDsignals may provide a way to diagnose neurological disorders that impactspecific circuits, as well as to screen for therapeutic agents to treatsuch disorders.

BRIEF SUMMARY OF THE INVENTION

The present disclosure is directed to apparatuses and methods involvingthe use of magnetic resonance imaging. Aspects of the disclosure includemodifying a target neural cell population in a first region of a brainto express light-responsive molecules. Using a light pulse, thelight-responsive molecules in the target neural cell population arestimulated. Multiple regions of the brain are scanned via magneticresonance imaging. The scans allow for observation of a neural reactionin response to the stimulation in at least one of the multiple regionsof the brain. In response to the observations, a determination is madewhether neural projection in a second region of the brain are connectedto at least some of the cells in the modified target cell population inthe first region of the brain.

Aspects of the present disclosure relate generally to exciting and/orinhibiting neural cells in vivo using light and mapping of the neuralresponse, by employing magnetic resonance imaging and methods relatingto the optogenetic modification of cells. In a more specific embodiment,functional magnetic resonance imaging (fMRI) using blood oxygenationlevel-dependent (BOLD) signals is used to map neural responses.

Certain aspects of the present disclosure relate to integratinghigh-field fMRI output with optogenetic stimulation of cells. Alight-activated, light-responsive molecule, for example an opsin, isintroduced into specifically targeted cell types and circuit elementsusing cell type-specific promoters to allow millisecond scale targetedactivity modulation in vivo. An opsin is light-activated and regulatesthe transmembrane conductance of a cell that expresses the opsin. Theopsin can be a single component microbial light-activated conductanceregulator. The genetic material of a desired opsin is modified toinclude cell type-specific promoters as well as promoters allowing foroptimal expression in the animal. The opsin can be modified to expressin mammalian cells, for example.

Other aspects of the present disclosure are directed to apparatuses andmethods involving the modification of a target neural cell population ina first region of a brain to express light-responsive molecules.Following modification, the light-responsive molecules are stimulated inthe target neural cell population by using a light pulse. While thetarget neural cell population is being stimulated, multiple regions ofthe brain are scanned using an fMRI machine. The fMRI scans are used toobserve neural reaction in response to the stimulation in at least oneof the multiple regions of the brain and to determine therefrom thenetwork communication characteristics relating to, but is notnecessarily the same as, the anatomical neural projection pattern.

Other aspects of the present disclosure are directed to apparatuses andmethods involving verifying BOLD responses. The method includesmodifying a target neural cell population to express light-responsivemolecules in a first region of a brain. The light-responsive moleculesexcite the target cell population in response to light. Thelight-responsive molecules in the target neural cell population arestimulated using a light pulse. At least the first region of the brainis scanned with an fMRI machine during light stimulation of the targetneural cell population. Based at least in part on a BOLD signal responsein the target neural cell population due to light stimulation, a BOLDsignal response is assessed from an electronic stimulation in the targetneural cell population.

In some embodiments, modifying neural cells may comprise delivering alight-responsive molecule (e.g., ChR2) to neural cells of a first brainregion. The neural cells of the first brain region may be stimulated bypositioning an optical fiber at, and applying light pulses to, the firstbrain region. Multiple regions of the brain may be scanned by acquiringmagnetic resonance images of first and second brain regions to identifythe neural cells of the second brain region that are connected to theneural cells of the first brain region.

In other embodiments, the neural cells of the first brain region may bestimulated by positioning optical fiber at, and applying light pulsesto, the second brain region. Multiple regions of the brain may bescanned by acquiring magnetic resonance images of first and second brainregions to identify the neural cells of the first brain region that areconnected to the neural cells of the second brain region.

In some embodiments, the first brain region may be in the motor cortexand the second brain region may be in the thalamus, or vice versa. Inother variations, the first brain region may be the anterior or theposterior thalamus. In some embodiments, the first brain region may bein the thalamus and the second brain region may be in the somatosensorycortex. Scanning multiple regions of the brain may comprise acquiringmagnetic resonance images of bilateral regions of the somatosensorycortex and/or motor cortex.

Various embodiments, relating to and/or using such methodology andapparatuses, can be appreciated by the skilled artisan, particularly inview of the figures and/or the following discussion.

The above overview is not intended to describe each illustratedembodiment or every implementation of the present disclosure. While thedisclosure is amenable to various modifications and alternative forms,specifics thereof have been shown by way of example in the drawings andwill be described in detail. It should be understood, however, that theintention is not to limit the disclosure to the particular embodimentsdescribed. On the contrary, the intention is to cover all modifications,equivalents, and alternatives falling within the scope of the disclosureincluding aspects defined in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Various example embodiments may be more completely understood inconsideration of the following detailed description in connection withthe accompanying drawings, in which:

FIG. 1A shows a brain subjected to an optogenetic fMRI (“ofMRI”), inaccordance with an example embodiment of the present disclosure.

FIG. 1B shows the response of the excited cells in FIG. 1A, inaccordance with an example embodiment of the present disclosure.

FIG. 2A shows a brain subjected to an ofMRI, in accordance with anexample embodiment of the present disclosure.

FIG. 2B shows the response of the excited cells in FIG. 2A, inaccordance with an example embodiment of the present disclosure.

FIG. 3A shows an fMRI of neurons modified to react to light, inaccordance with an example embodiment of the present disclosure.

FIG. 3B shows the response of the cells in FIG. 3A, in accordance withan example embodiment of the present disclosure.

FIG. 4 shows the BOLD and ofMRI-HRF (haemo-dynamic) responses of atarget cell population, in accordance with an example embodiment of thepresent disclosure. ofMRI haemodynamic response (averaged acrossactivated voxels in motor cortex) during 20 s (top) and 30 s (bottom)optical stimuli (Left) is shown and also depicted are mean over stimulusrepetitions; baseline, mean pre-stimulation signal (Right). Top panels,n=3; bottom panels, n=8.

FIG. 5 depicts one variation of a system and method for delivering aviral vector to a neuronal population for optogenetic stimulation andfMRI analysis. FIG. 5 a shows transduced cell, represented by triangles,and the light and location (1 . . . 9) of coronal slices, indicated bydots. FIG. 5 b shows confocal images of ChR2-EYFP expression in M1(left); higher magnification (right). FIG. 5 c shows optrode recordingsduring 473 nm optical stimulation (20 Hz/15 ms pulse width); spiking issignificantly elevated (error bar indicates±s.d., two-sample t-test; ***indicates P<0.001; n=3). “Pre” indicates spike frequencypre-stimulation; “Stim” indicates spike frequency during stimulation;“Post” indicates spike frequency post-stimulation. FIG. 5 d shows BOLDactivation observed with AAV5-CaMKIIα::ChR2-EYFP but not with salineinjection (P<0.001; asterisk, optical stimulation).

FIG. 6 depicts nonlocal mapping of the casual role of cells defined bylocation and genetic identify, in accordance with an example embodimentof the present disclosure. FIG. 6 a schematically depicts a variation ofa system and method for AAV5-CaMKIIα::ChR2-EYFP injection and opticalstimulation in M1. Slices in “c”: ‘1’ and ‘2.’ FIG. 6 b showsfluorescence/bright-field images of ChR2-EYFP in thalamus (left); theconfocal image (right) shows that expression is limited to axons. FIG. 6c depicts slices ‘1’ and ‘2’ that were taken at points ‘1’ and ‘2’ ofFIG. 3 a and shows thalamic ofMRI during M1 optical stimulation (top);superimposed on the Paxinos atlas (bottom). FIG. 6 d are plots thatsummarize ofMRI-HRF results. FIG. 6 e schematically depicts a M1 optrodeand a thalamic electrode. FIG. 6 f shows thalamic spiking that followsM1 optical stimulation; delay consistent with BOLD. FIG. 6 g depictstypical M1 and thalamus spikes that arise with M1 optical excitation.FIG. 6 h are histograms that summarize M1 and thalamus spiking profiles(error bar indicates±s.d., two-sample t-test; *** indicates P<0.001;n=5). FIG. 6 i depicts spike-frequency time histograms.

FIG. 7 depicts control of cells defined by location, genetic identity,and wiring during ofMRI, in accordance with an example embodiment of thepresent disclosure. FIG. 7 a schematically depicts a variation of asystem and method for M1 injection of AAV5-CaMKIIα::ChR2-EYFP andoptical stimulation of the thalamus. Coronal slices shown in FIG. 7 cmarked as ‘1 . . . 6’ and ‘7 . . . 12’. FIG. 7 b shows a ChR2 expressionpattern confirming expression in cortical neurons (left) andcortico-thalamic projections (right; see also Supplementary FIG. 5 of“Global and local fMRI signals driven by neurons defined optogeneticallyby type and wiring” Nature, Vol. 465, 10 Jun. 2010, pp, 788-792). FIG. 7c shows BOLD ofMRI data obtained in thalamus (above) and cortex (below).FIG. 7 d depicts plots of ofMRI-HRF for cortical (grey) and thalamic(black) BOLD signals elicited by optical stimulation of cortico-thalamicfibers in thalamus. Both ofMRI-HRFs ramp slowly by comparison withintracortical results in FIG. 5.

FIG. 8 shows recruitment of bilateral cortices by the anterior thalamus,in accordance with an example embodiment of the present disclosure. FIG.8 a schematically depicts a variation of a system and method forthalamic injection of AAV5-CaMKIIα::ChR2-EYFP and posterior/anterioroptical stimulation. Coronal slices marked ‘A1 . . . A12’ and ‘B1 . . .B12’. FIG. 8 b depicts an image where fluorescence is overlaid ontobright-field (left) and confocal image (right) illustrating transductionin the thalamus (left) and cortical projections in the internal andexternal capsule (right). FIG. 8 c depicts scans of posterior thalamusstimulation-evoked ofMRl signal in the ipsilateral thalamus andsomatosensory cortex. FIG. 8 d is a plot of ofMRI-HRFs. Excited volumes:5.5±1.3 mm³ (thalamus); 8.6±2.5 mm³ (somatosensory cortex) (n=3). FIG. 8e depicts scans of anterior thalamus stimulation-evoked ofMRI signal inthe ipsilateral thalamus and bilateral motor cortex. FIG. 8 f is a plotof ofMRI-HRFs. Excited volumes: 1.5 mm3 (thalamus); 10.1 mm3(ipsilateral cortex); 3.7 mm3 (contralateral cortex).

DETAILED DESCRIPTION AND EXAMPLE EMBODIMENTS

Aspects of the present disclosure may be more completely understood inconsideration of the detailed description of various embodiments of thepresent disclosure that follows in connection with the accompanyingdrawings. This description and the various embodiments are presented byway of the Examples and in “Global and local fMRI signals driven byneurons defined optogenetically by type and wiring,” Nature, Vol. 465,10 Jun. 2010, pp, 788-792, which is hereby incorporated by reference inits entirety. The embodiments and specific applications discussed hereinmay be implemented in connection with one or the above describedaspects, embodiments and implementations, as well as those shown in thefigures and described below. Reference may also be made to the followingbackground publications: in U.S. Published Patent Application No.2010/0190229, entitled “System for Optical Stimulation of Target Cells”to Zhang et al.; U.S. Published Patent Application No. 2010/0145418,also entitled “System for Optical Stimulation of Target Cells” to Zhanget al.; and U.S. Published Patent Application No. 2007/0261127, entitled“System for Optical Stimulation of Target Cells” to Boyden et al. Theseapplications form part of the provisional patent document and are allhereby incorporated by reference in their entirety. As is apparent fromthese publications, numerous opsins can be used in mammalian cells invivo and in vitro for provide optical stimulation and control of targetcells. For example, when ChR2 is introduced into a cell, lightactivation of the ChR2 channelrhodopsin results in excitation and firingof the cell. In instances when NpHR is introduced into a cell, lightactivation of the NpHR opsin results in inhibition of the cell. Theseand other aspects of the disclosures of the above references patentapplications may be useful in implementing various aspects of thepresent disclosure.

In certain embodiments of the present disclosure, a viral vectorincluding a light sensitive molecule is injected in cells in the primarymotor cortex of an animal. The viral vector infects a chosen cell type,for example cortical neurons, while not infecting surrounding cells ofdifferent cell types. A cannula is implanted into the animal's brain toallow access for both the injection of the virus and for an opticalfiber to provide light to the infected cells. The cannula, opticalfiber, and any other accessories are fabricated from magneticresonance-compatible materials in order to minimize susceptibilityartifact during MRI scanning. Light pulses are provided by the opticalfiber to the cells that have been infected in the motor cortex. The wavelength of the light is chosen based on the light-sensitive moleculeintroduced into the cell population. Light pulses are delivered to theneurons expressing the light-sensitive molecules. In response, anoptically evoked BOLD signal is observed in the cortical grey matter atthe virus injection and optical stimulation site. The BOLD signal isobserved in fMRI slices of the motor cortex. Additional fMRI slicescapture downstream responses during optical stimulation of the corticalneurons. The additional fMRI slices are centered on the thalamus, forexample. The stimulation of the infected cells in the motor cortexresults in cortico-thalamic axonal projection fibers being observed. Areaction is observed in the thalamus despite the fact that no cells inthe thalamus have been infected with light sensitive molecules.

BOLD fMRI is a technology for non-invasive, whole-brain imaging. Boldsignals reflect complex and incompletely understood changes in cerebralblood flow (CBF), Cerebral blood volume (CBV), and cerebral metabolicrate of oxygen consumption (CMRO2) following neuronal activity. For avariety of reasons, it is useful and important to understand what kindsof activity are capable of triggering BOLD responses.

In various embodiments of the present disclosure, the local stimulationof the cortex during fMRI is used to determine if unidirectionallytriggered BOLD responses are being observed and measured. In thisembodiment, antidromic drive found in electrical stimulation iseliminated, and allows for global causal connectivity mapping. Robustthalamic BOLD signals are observed in response to cortex stimulation.The properties of the thalamic response are distinct from the responsein the cortex. For example, the thalamic response is delayed in time ascompared to the response observed in the cortex.

In certain more specific embodiments, fMRI scanning occurs several days(e.g., in some instances, at least 10 days) after virus injection. fMRIsignals are acquired in 0.5 mm coronal slices. To assess the neuralresponse at the site of the injection, the slices are centered aroundthe motor cortex. To assess the neural response at areas remote from themotor cortex 0.5 mm slices are acquired in the area of interest. A 473nm light pulse is delivered by an optical fiber at a rate of 20 Hz, witha 15 ms pulse width to the targeted cells expressing thelight-responsive molecules.

In another embodiment consistent with the present disclosure, a viralvector including a light sensitive molecule is injected in cells in theprimary motor cortex of an animal. The viral vector delivers alight-responsive molecule to a chosen cell type, for example corticalneurons, while not infecting surrounding cells of different cell types.A cannula is implanted at a second location remote from the corticalneurons expressing the light-responsive molecule. The viral vector isinjection into the motor cortex, and the cannula is implanted in thethalamus, for example. Providing light to the thalamus allows forconfirmation of the functional projection patterns in the brain. Thelight-responsive molecules trigger spikes in illuminated photosensitiveaxons that both drive optical synaptic output and back-propagate throughthe axon to some of the stimulated cells. This permits optical fMRImapping during selective control of the motor cortical cells thatproject to the thalamus. Robust BOLD signals are observed both locallyin the thalamus and in the motor cortex. This result is consistent withthe recruitment of the topologically targeted cells both locally anddistally. It also demonstrates that stimulation of the axons of theneurons expressing the light-responsive molecules is sufficient toelicit BOLD responses in remote areas. This also illustrates thefeasibility of in vivo mapping of the global impact of cells defined notonly by anatomical location of the body of the cell and the geneticidentity, but also the connection topology. The projections of theinfected cells can be mapped based on the reaction of the axons to lightstimulation at areas remote from the body of the cell.

In certain embodiments consistent with the present disclosure, a viralvector carrying a light-responsive molecule is injected into cells inthe thalamus. A cannula is implanted in order to deliver light to thetarget cell population expressing the light-responsive molecules in thethalamus. Light is delivered to the target cells. fMRI scans of thethalamus as well as other areas of the brain allow functional mapping ofthe thalamic projections to the motor cortex. Scanning other regions ofthe brain as well can show responses in the sensory cortex or otherparts of the brain as well. Because motor control and planning involvebilateral coordination, mapping of thalamic projections is more likelyto involve both ipsilateral and contralateral pathways. The use of lightstimulation of thalamic nuclei allows isolation of functional mapping ofthalamocortical projections without also showing cortical-thalamicprojections.

In certain more specific embodiments, it is shown that opticalstimulation of posterior thalamic nuclei resulted in a strong BOLDresponse, both at the site of stimulation and in the posterioripsilateral somatosensory cortex. Optically stimulating excitory cellbodies and fibers in anterior thalamic nuclei resulted in BOLD responseat the site of stimulation and also ipsilateral and contralateralcortical BOLD responses consistent with the bilaterality of anteriorthalamocortical nuclei involvement in motor control and coordination.

Turning to FIG. 1A, a section of a brain 100 is depicted. The brain 100includes two regions, separated from each other, with cell populationsof interest. The first target cell population 102 is modified to includea light-responsive molecule. The second cell population 110 is connectedto the first target cell population 102 through a neural projection 116.An fMRI compatible cannula 106 is implanted and an optical fiber 108 isdelivered through the cannula 106 to the target cell population 102. Theoptical fiber 108 provides light 104 to the light-responsive moleculesin the target cell population 102. In response to the light delivery,the light-responsive molecules in the target cell population 102 areexcited and the excitation spreads down the neural projection 116 to thecell population 110 in a second region of the brain. The progress of theexcitation of the target cell population 102 and the remote cellpopulation 110 is captured using an fMRI machine 112. The fMRI machinescans the brain 100 at designated areas. The results 114 of the fMRIscan show evidence of excitation in the remote cell population 110.

In certain embodiments the target cell population 102 is injected with avirus at the same cite as the cannula. The virus injection includes aviral vector virus to deliver a light-responsive molecule, such aschannelrhodopsin (ChR2) to the target cell population. The viral vectorincludes promoters to drive expression of the ChR2 in the target cellpopulation. The promoters can be chosen based on the target cellpopulation 102 so that expression of ChR2 is limited to the desired celltype. For example, in adult rats an adeno-associated viral vectorAAV5-CaMKIIα::ChR2(H134R)-EYFP can be used to drive expression of a ChR2specifically in Ca²⁺/calmodulin-dependent protein kinase IIα(CaMKIIα)-expressing principal cortical neurons, but not in neighboringGABAergic or glial cells. In embodiments where ChR2 is thelight-responsive molecule, the optical fiber 108 provides a 473 nm lightpulse at 20 Hz. This has been found to reliably drive local neuronalfiring in vivo.

In more particular embodiments viral injection occurred at least 10 daysbefore the fMRI scans were taken.

In certain embodiments the target cell population 102 is located in themotor cortex (M1) and the remote cell population 110 is located in thethalamus.

The resultant images 114 shows the scan results for two slices, 1 and 2,in the thalamus. The images 114 depict the blood oxygenationlevel-dependent (BOLD) signals elicited in response to excitation of thetarget cell population 102 with light.

Turning to FIG. 1B, the responses of the target cell population 102 andthe remote cell population 110 are depicted. The top graph 120 shows arecording of the electrical response to light stimulation at thelocation of the target cell population 122 (labeled M1 recording) andthe electrical response of the remote cell population 124 (labeledthalamus recording) in response to light stimulation of the target cellpopulation 102. The excitation is shown to have occurred at the targetcell population 102 approximately 8 ms before it occurs at the remotecell population 110. The reading depicted in graph 120 was obtained byintroducing electrodes at M1 and at the thalamus to verify that thetarget cell population 102 and the remote cell population 110 respond tothe light 104.

Graph 130 depicts the BOLD signal change in response to the lightstimulation both at the cortex (the location of the target cellpopulation 102) and at the thalamus (the location of the remote cellpopulation 110). The cortex response 132 initiates earlier than thethalamus response 134, consistent with the delay found in graph 120.

Turning to FIG. 2A, a method consistent with an embodiment of thepresent disclosure is depicted. A virus containing a sequence forexpressing a light-responsive molecule, such as ChR2, is injected intotarget cell population 202. A cannula 206 is implanted in a secondregion of the brain so that optical fiber 208 can provide light 204 toremote cell population 210. The light at the remote location excites theaxon projections 216 of the cells 202 that connect to the cells in theremote location. The axon projections 218 of the remote cell population210 also potentially connect the two cell populations. This results in aBOLD response both at the target cells 202 and the remote cells 210.This also permits functional mapping of the cells in the two areas. AnMRI machine 212 is used to collect fMRI scans 220 showing a BOLDresponse both at the injection site (slices 1-6) and at the site ofstimulation (slices 7-12).

In certain more specific embodiments, the virus injection site is in thecortex and the light stimulation is provided to the thalamus. FIG. 2Bdepicts the percentage of BOLD signal change in response to thestimulation. As shown in the figure, the time delay between the responseat the thalamus and the motor cortex is lessened. In addition, the BOLDresponse ramps up more slowly in both the thalamus and the cortex inthis embodiment. The fMRI scans 220, along with the BOLD responses 232and 234 of FIG. 2B indicate that the axon projections 218 were notactivated and the remote cell population 210 was not excited by thelight, while the axon projection 216 was excited.

Consistent with an embodiment of the present disclosure, FIG. 3A depictsa scan series wherein a viral vector is injected into the thalamus 310.The viral vector is cell type specific and introduces a light-responsivemolecule into specific cells of the thalamus. A cannula provides accessto the injection site for an optical fiber. The optical fiber is used toexcite the thalamic nuclei that have been infected with thelight-responsive molecules. The optical fiber is used to provide lightto either posterior thalamic nuclei (a) or anterior thalamic nuclei (b).Projection 318 extends from group A to cells within a remote cellpopulation 302. Projection 316 extends from group B in the thalamicnuclei to remote cell population 302. In certain specific embodiments,remote cell population 302 is located in the motor cortex. Scanning thebrain during stimulation by fMRI 312 results in coronal slices 320 beingobtained when the posterior thalamic nuclei are excited and slices 322when the anterior thalamic nuclei are excited.

FIG. 3B depicts graphs of the percentage of signal change in the BOLDresponse. Graph 330 corresponds to excitation of the posterior thalamus.Graph 332 corresponds to excitation of the anterior thalamus. Coronalslices 322 and Graph 332 indicate that excitation of anterior thalamicnuclei results in excitation in the thalamus (at the site light isprovided), and both the ipsilateral cortex and the contralateral cortex.This is consistent with the anterior thalamus being significant in motorcontrol and coordination.

The above embodiments can be used individually or together to providefunctional mapping of the brain. In certain embodiments both the motorcortex and the thalamus are infected with light-responsive moleculesthat respond to different wavelengths of light. This allows for forwardand backward mapping of the connections between cells in the cortex andthe thalamus. Further, light of one wavelength can be provided at a sitein the thalamus to excite the axon projections of the target cellpopulation in the motor cortex to provide a map of the connectionsbetween the target cell population and cells in the thalamus. Light of asecond wavelength can be used to stimulate thalamic cells and determinefunctional connections of the thalamus cells based on the axonprojections of the thalamic cells. The thalamic cells infected with thelight-responsive molecules responsive to a second wavelength of lightcan be the same cells that were activated by the motor cortex infectedcells. In an alternative embodiment the thalamic cells infected can bedifferent cells.

Certain embodiments consistent with the present disclosure can be usefulfor determining the progression of a degenerative disease. fMRI scansobtained over time can be compared to determine the presence ofdeteriorating function. The fMRI scans can also be used to determine ifnew connections are being made in response to damage to previousconnections.

In certain embodiments the fMRI scans can be used to determine theeffectiveness of a drug. For example, fMRI scans can be taken before andafter administration of a drug intended to alter the functionalresponsive of a target cell population. The scans can be compared todetermine whether the drug has produced the intended result, and if not,the dose of the drug can be adjusted based on the observations of thefMRI scans.

In certain embodiments of the present disclosure, the results of thefMRI scans are used to confirm the trigger of the BOLD response depictedin the fMRI slices. In such embodiments, light pulses are delivered totargeted neurons. The BOLD signals from a fMRI scan of the neurons,after they have been infected with a light-responsive molecule while alight pulse is being delivered to the neurons, is compared to a fMRIscan of the same neurons prior to infection with the light pulse beingdelivered. In such instances no detectable BOLD signal could be elicitedfrom the neurons prior to infection. However, after infection a robustBOLD signal was observed in response to the light pulse. Further, theBOLD dynamics observed by optically driving the cell populationexpressing the light-responsive molecule match the dynamics ofconventional stimulus-evoked BOLD-fMRI. In particular, as depicted inFIG. 4, the optogenetic fMRI haemodynamic response function (ofMRI-HRF)signal onset occurred after 3 seconds but within 6 seconds of stimulusonset. Likewise, offset was reflected by a drop in BOLD signal contrastbeginning within 6 seconds and returning to baseline in approximately 20seconds after optical stimulation. The pronounced post-stimulusundershoot observed during systemic somatosensory stimulation in humansand animals was observed in ofMRI-HRFs as well. All of these dynamicproperties derived from driving a defined, specific cell population,correspond closely to previous measurements on conventionalsensory-evoked BOLD.

In various embodiments consistent with the present disclosure, neuronsare infected with light-responsive molecules as discussed with respectto FIGS. 1A, 2A, and 3A. The activation of the light-responsivemolecules can be used to determine the correlation between neuron firingand observed responses for a variety of neural scanning devices orprocedures. The scanning includes, but is not limited to: MRI, computedtomography (CT), electroencephalography (EEG), and positron emissiontomography (PET).

In certain embodiments more than one type of a light-responsive moleculeis introduced into the brain. The light-responsive molecule can be oneof several opsins and channelrhodopsins including, but not limited to,VChR1, optoXRs, SFOs, and ChR2 for excitation/modulation of cell inresponse to specific wavelengths of light, and NpHR, BR, AR, and GtR3for inhibition of cells in response to specific wavelengths of light. Incertain embodiments, the light-responsive molecule is introduced intothe brain by a viral vector (such as an AAV) encoding thelight-responsive molecule.

EXAMPLES

Despite a rapidly-growing scientific and clinical brain imagingliterature based on functional magnetic resonance imaging (fMRI) usingblood oxygenation level-dependent (BOLD)′ signals, it remainscontroversial whether BOLD signals in a particular region can be causedby activation of local excitatory neurons. This difficult question iscentral to the interpretation and utility of BOLD, with majorsignificance for fMRI studies in basic research and clinicalapplications. Using a novel integrated technology unifying optogeneticcontrol of inputs with high-field fMRI signal readouts, we show herethat specific stimulation of local CaMKIIn-expressing excitatoryneurons, either in the neocortex or thalamus, elicits positive BOLDsignals at the stimulus location with classical kinetics. We also showthat optogenetic fMRI (ofMRI) allows visualization of the causal effectsof specific cell types defined not only by genetic identity and cellbody location, but also by axonal projection target. Finally, we showthat ofMRI within the living and intact mammalian brain reveals BOLDsignals in downstream targets distant from the stimulus, indicating thatthis approach can be used to map the global effects of controlling alocal cell population In this respect, unlike both conventional fMRIstudies based on correlations and fMRI with electrical stimulation thatwill also directly drive afferent and nearby axons, this ofMRI approachprovides causal information about the global circuits recruited bydefined local neuronal activity patterns. Together these findingsprovide an empirical foundation for the widely-used fMRI BOLD signal,and the features of ofMRI define a potent tool that may be suitable forfunctional circuit analysis as well as global phenotyping ofdysfunctional circuitry.

Blood oxygenation level-dependent functional magnetic resonance imaging(BOLD fMRI) is a widely used technology for non-invasive whole brainimaging. BOLD signals reflect complex and incompletely understoodchanges in cerebral blood flow (CBF), cerebral blood volume (CBV), andcerebral metabolic rate of oxygen consumption (CMR0₂) following neuronalactivity. Candidate circuit elements for triggering various kinds ofBOLD signals include excitatory neurons, mixed neuronal populations,astroglia, and axonal tracts or fibres of passage. Importantly, it isnot clear which kinds of activity are capable of triggering BOLDresponses, placing limitations on interpretation for both clinical andscientific applications. For example, it is sometimes assumed thatpositive BOLD signals can be triggered by increased activity of localexcitatory neurons, but this remains to be shown empirically, achallenge that seriously confounds fMRI interpretation. Moreover, theuse of MRI-compatible electrodes for local stimulation, although ofpioneering significance, will nevertheless drive all local excitatory,inhibitory, and modulatory cell types, as well as antidromically drivenon-local cells that happen to have axons within the stimulated region,thereby confounding functional circuit mapping using BOLD. We sought toaddress these challenges by integrating high-field fMRI output withoptogenetic stimulation, in which single-component microbiallight-activated transmembrane conductance regulators are introduced intospecifically targeted cell types and circuit elements'” using celltype-specific promoters to allow millisecond-scale targeted activitymodulation in vivo.

Materials and Methods

Virus-Mediated Opsin Expression

In adult rats, the primary motor cortex (M1) was injected with theadeno-associated viral vector AAV5-CaMKIIα::ChR2(H134R)-EYFP to driveexpression of a channelrhodopsin (ChR2) specifically inCa2+/calmodulin-dependent protein kinase II α (CaMKIIα)-expressingprincipal cortical neurons, but not in GABAergic or glial cells. ThepAAV-CaMKIIα-hChR2(H134R)-EYFP plasmid was designed and constructed bystandard methods and packaged as AAV5. The pAAV-CaMKIIα-ChR2(H134R)-EYFPplasmid was constructed by cloning CaMKIIα-ChR2(H134R)-EYFP into an AAVbackbone using MluI and EcoRI restriction sites. The recombinant AAVvectors were serotyped with AAV5 coat proteins and packaged by the viralvector core at the University of North Carolina; titers were 2×10¹²particles/mL for both viruses. The virus was stereotaxically injectedand cannulas placed at the locations where optical stimulation wasplanned. Concentrated virus was delivered using a 10-μl syringe and34-gauge needle; volume and flow rate (0.1 μl min⁻¹) were controlled byinjection pump. Maps and clones are available at www.optogenetics.org.

Female adult (>10 weeks old) Fischer and Sprague-Dawley (250-350 g) ratswere the subjects; animal husbandry and all aspects of experimentalmanipulation were in strict accord with guidelines from the NationalInstitute of Health and have been approved by members of the StanfordInstitutional Animal Care and Use Committee (IACUC). Rats wereanaesthetized using 1.5% isoflurane (for surgeries longer than 1 hr) ori.p. injection (90 mg/kg ketamine and 5 mg/kg xylazine). The top of theanimal's head was shaved, cleaned with 70% ethanol and betadine and thenpositioned in the stereotactic frame. Ophthalmic ointment was applied, amidline scalp incision was made, and small craniotomies were performedusing a drill mounted on the frame. Four types of surgeries wereconducted: I) viral injection (1 μl/site) and cannula (1.5 mmprojection) placement in M1 (+2.7 mm AP, +3.0 mm ML right hemisphere,two injections at −2.0 and −2.5 mm DV); II) 4 viral injections acrosscortex (1: +5.2 mm AP, +2.0 mm ML right hemisphere, one 2 μl injectionat −3.0 DV; 2: +3.2 mm AP, +3.5 mm ML right hemisphere, 3 injectionseach 0.7 μl at −3.5 mm, −3.0 mm, and −2.5 mm DV; 3: +2.7 mm AP; +0.5 mmML right hemisphere; 2 injections 1 μl each at −3.0 mm and −2.5 mm DV;4: −0.3 mm AP; +3.0 mm ML right hemisphere; 2 injections 1 μl each at−2.5 mm and −2.0 mm DV; and the cannula (6.5 mm projection) was placedin ventral thalamus at the border with ZI (−4.3 mm AP; +2 mm ML righthemisphere; −6.5 mm DV); III) viral injection (1 μl/site) and cannula(5.25 mm projection) placement in thalamus (+2.7 mm AP, +3.0 mm ML righthemisphere, two injections at −5.25 and −5.75 mm DV). IV) Doublefloxedinverted-open reading frame (DIO) ChR2-EYFP was injectedstereotactically into the motor cortex (2.0 mm AP; 1.42 mm ML, twoinjections at −1.25 mm and −1.75 mm DV) of 5-10-week-old transgenic miceexpressing Cre recombinase in fast-spiking parvalbumin expressingGABAergic interneurons (PV::Cre).

Concentrated virus was delivered using a 10 μl syringe and a thin 34gauge metal needle; injection volume and flow rate (0.1 μl/min) werecontrolled with an injection pump. After the final injection, the needlewas left in place for 5 additional minutes and then slowly withdrawn. AnMRI compatible cannula fiber guide (8IC313GPKXXC) was inserted throughthe craniotomy. One layer of adhesive cement followed by cranioplasticcement was used to secure the fiber guide system to the skull. After 10min, the scalp was sealed using tissue adhesive. The animal was kept ona heating pad during recovery from anesthesia. Buprenorphine (0.03mg/kg) was given subcutaneously following the surgical procedure tominimize discomfort. A dummy cannula (8IC312DCSPCC) was inserted to keepthe fiber guide patent.

To avoid scanning animals with damage associated with the cannulaimplantation, detailed anatomical MRI scans were performed to check fortissue damage. In cases where damage near the cannula was detected inthe T2 weighted high-resolution anatomical images, animals were rejectedand not used in experiments. Moreover, animals used for fMRI studieswere examined post-mortem for local invasion and for gliosis, using DAPIand GFAP staining. GFAP is a sensitive marker for gliosis and can reportchanges in local glial number, glial activation, and inflammation. Inall subjects used for fMRI, we did not see evidence for cellularinvasion or for gliotic changes (See Supplementary FIG. 1 of “Global andlocal fMRI signals driven by neurons defined optogenetically by type andwiring,” Nature, Vol. 465, 10 Jun. 2010, pp, 788-792) beyond theexpected 30-50 μm from the cannula, indicating that the boundaries ofthe BOLD responses are not determined by local damage or gliosis underthese conditions.

Opsin Expression Validation

To validate specificity, sensitivity and spatial distribution of opsinexpression, brain slices were prepared for optical microscopy andimmunohistochemistry. Coronal sections (40-μm thick) were cut on afreezing microtome and stored in cryoprotectant (25% glycerol, 30%ethylene glycol, in PBS) at 4° C. until processed forimmunohistochemistry. Confocal fluorescence images were acquired on ascanning laser microscope using a 20×/0.70NA or a 40×/1.25NA oilimmersion objective, while large field images of entire slices werecollected on a Leica MZ16FA stereomicroscope.

Immunohistochemistry

To verify the phenotype of cells, rodents were anaesthetized with 65mg/kg sodium pentobarbital and transcardially perfused with ice-cold 4%paraformaldehyde (PFA) in PBS (pH 7.4). Brains were fixed overnight in4% PFA and then equilibrated in 30% sucrose in PBS. 40 μm-thick coronalsections were cut on a freezing microtome and stored in cryoprotectant(25% glycerol, 30% ethylene glycol, in PBS) at 4° C. until processed forimmunohistochemistry. Free-floating sections were washed in PBS and thenincubated for 30 min in 0.2% Triton X-100 (Tx100) and 2% normal donkeyserum (NDS). Slices were incubated overnight with primary antibody in 2%NDS (Mouse anti-CaMKIIα 1:500, Abcam, Cambridge, Mass.; Mouseanti-Parvalbumin 1:500, Sigma, St Louis, Mo.; Rabbit anti-GABA 1:500,Millipore, Billerica, Mass.; Chicken anti-GFAP 1:250, Millipore; Mouseanti-MAP2 1:500, Sigma). Sections were then washed with PBS andincubated for 2 hr at RT with secondary antibodies (Donkey anti-Mouseconjugated to either Cy3 or FITC, donkey anti-Rabbit Cy5 and donkey-antichicken Cy5, all 1:1000, Jackson Laboratories, West Grove, Pa.). Sliceswere then washed, incubated with DAPI (1:50,000) for 20 min, washedagain, and mounted on slides with PVA-Dabco (Sigma). Confocalfluorescence images were acquired on a scanning laser microscope using a20×/0.70NA or a 40×/1.25NA oil immersion objective.

In Vivo Optical Stimulation

20 Hz, 15 ms pulsewidth stimulation with 473 nm light was used for allfMRT and optrode recordings. 300 μm diameter optical fibers were usedwith the optical fiber output power level at approximately 6 mW. Thesepower levels correspond to 85 mW mm⁻² at the fiber output, but more than10-fold less over the majority of the excitation volume given theexpected light scattering profile. Assuming 1 mW/mm² is the minimumlight power needed to activate ChR2, the light penetration depth ofdirect light activation is expected to be ˜0.95 mm. Optical stimulationpower must be set with care in order to avoid potential BOLD signalconfound due to heating; we have found that at higher laser power levelsor with steady illumination, laser synchronized signal intensity changecan be observed even in control animals; the BOLD sequence, which givesT₂*-weighting has been found to have no significant temperaturedependence at lower temperatures, while high enough temperature causingtissue damage has been found to result in signal amplitude decrease.Therefore, it was decided to use ≦˜6 mW of laser power and maintainedpulsed waveforms with 30% duty cycle.

Analysis of Electrophysiological Data

Threshold search in Clampfit was used for automated detection of spikesin multi-unit recording, which was then validated by visual inspection.For traces with multiple spike populations, thresholds were set tocapture all the spikes; during bursting, it is likely that multipleneurons were recorded from simultaneously.

Optogenetic fMRI

Rodent subjects were connected to the optical fiber and ventilator (1.3%isoflurane), physiological monitoring systems and radio-frequency coil,and placed in the magnetic resonance-compatible stereotaxic frame. Aftersubject placement in the scanner, blue (473 nm) light pulsed at 20 Hz(15 ms pulse width) was periodically applied through the optical fiberat 1 min intervals while repeated BOLD scans of large brain volumes wereconducted.

fMRI scans were conducted with a small animal dedicated MRI scanner,custom designed pulse sequences, RF coils, and cradle. The small animalscanner consisted of a Magnex scientific superconducting magnet with 7.0Tesla (T) field strength, RRI gradient with clear bore size of 9 cm,maximum gradient amplitude of 770 mT/m and maximum slew rate of 2500T/m/s and a General Electric (GE) console and radiofrequency (RF)amplifiers with maximum RF amplitude of 24.7 μT. The animals were firstanesthetized in a knockdown box with 4% isoflurane. After approximately5 minutes in the knockdown box, the animal was intubated, placed on acustom-designed MRI-compatible cradle with a stereotaxic frame, and thetracheal tube connected to a ventilator (Harvard Apparatus, Model 683Small Animal Ventilator) with 1.3-1.5% isoflurane, 35% O₂, 65% N₂O inputgas and a capnometer (SurgiVet V9004). A 3.5 cm diameter custom-designedtransmit/receive single-loop surface coil was placed on the top of thetarget, and a 300 μm diameter optical fiber was then inserted throughthe guide. A fiber-optic rectal temperature probe was inserted and thecradle with the animal was inserted to the isocenter of the magnet.Expiratory CO2 content was continuously monitored by a capnometer. Theventilation volume and frequency (3.0-3.5 cc/stroke, 50-60 stroke/min)was controlled to keep the endtidal CO₂ level at ˜3.5% throughout thescan. Heated air was pumped into the bore to maintain animal's bodytemperature at physiological levels (34-38° C.).

fMRI scans were performed using conventional GRE-BOLD fMRI methods andpassband b-SSFP fMRI4 methods. Passband bSSFP-fMRI was designed to be a3D volumetric, b-SSFP sequence with stack-of-spirals readout trajectory.To get good slab selection for the passband bSSFP-fMRI scans, atime-bandwidth (TBW) of 12 pulse was designed with a duration of 1 ms.10 interleave in-plane spirals with 32 stack locations, 9.372 ms TR, 2ms TE resulted in 30 slices (2 slices discarded due to 3D slicedirection excitation profile roll-off margin) and 1.5 cm slice directionvolume coverage.

fMRI data was first reconstructed through a 2-dimensional (2D) and 3Dgridding reconstruction methods. The reconstructed 4D magnitude imagedata was then analyzed by calculating the individual voxel coherencevalue (c), defined as the magnitude of the frequency component ofinterest (|F(f₀)|) divided by the sum-of-squares of all frequencycomponents:√{square root over (Σ_(f)|F(f)|²)}; F:

(Fourier transform of temporal signal intensity; f₀: frequency ofstimulation—here, 1/60 Hz). Therefore, the coherence value (c) isbetween 0 and 1. The coherence value was thresholded at 0.35, colorcoded and overlaid onto T₂ anatomical images:

$c = \frac{{F\left( f_{0} \right)}}{\sqrt{\sum\limits_{f}^{\;}\;{{F(f)}}^{2}}}$

Coherence values (c) can be converted to z- and p-values given the mean(m), variance (σ²) of the null-hypotheses distribution. The followingformula can be used to calculate the corresponding z value given the cvalue, m, σ, and N (sample size for estimation of m and σ).

$z = {\frac{1}{\sigma}\left( {\sqrt{\frac{c^{2}}{\left( {1 - c^{2}} \right)}\left( {{\left( {N - 1} \right)\sigma^{2}} + {Nm}^{2}} \right)} - m} \right)}$

The p-value can then be estimated with an assumption for thenull-hypothesis. For example, in our study, coherence of 0.35corresponds to z-value of approximately 4.6, which gives a p-value ofapproximately 0.000002 when Gaussian distribution is assumed. Therefore,the p-value threshold in all our experiments can be assumed to be lessthan 0.001. For most of the data, the thresholded coherence value wasoverlaid onto T₂ anatomical images to show “activated” voxels. However,for the PV::Cre stimulation result, since pixels with opposite phasewith respect to stimulation were present, color-coded phase values ofpixels with coherence level over 0.35 were displayed to show thedistribution of positive and negative BOLD. The phase value (θ) wascalculated as the phase of the frequency component of interest,resulting in phase values between 0 and 2π (0 corresponds to no delaywith respect to stimulus, and π corresponds to the half cycle delay of30 s in these experiments).θ=∠(F(f ₀))

Voxel-based frequency analysis was used as the method with fewestassumptions instead of model-based methods since the HRF of ofMRI wasnot known.

In Vivo Recording and Analysis

After ofMRI, simultaneous optical stimulation and electrical recordingin living rodents was conducted using an optrode composed of anextracellular tungsten electrode (1MΩ, ˜125 μm) attached to an opticalfiber (˜200 mm) with the tip of the electrode deeper than the tip of thefiber to ensure illumination of the recorded neurons.

Simultaneous optical stimulation and electrical recording in livingrodents was conducted as described previously using an optrode composedof an extracellular tungsten electrode (1 MΩ, ˜125 μm) attached to anoptical fiber (μ200 μm) with the tip of the electrode deeper (˜0.4 mm)than the tip of the fiber to ensure illumination of the recordedneurons. For stimulation and recording in two distinct regions (M1 andthalamus), small craniotomies were created above both target regions.The optical fiber was coupled to a 473 nm laser diode from CrystaLaser.Optrode recordings were conducted in rats anesthetized with 1.5%isoflurane. pClamp 10 and a Digidata 1322A board were used to bothcollect data and generate light pulses through the fiber. The recordedsignal was bandpass filtered at 300 Hz low/5 kHz high (1800Microelectrode AC Amplifier) and filtered in Clampfit to remove 60 Hznoise. For precise placement of the fiber/electrode pair, stereotacticinstrumentation was used.

Results

In these experiments, the cortical virus injection site was also used asthe optical stimulation site for BOLD and electrophysiologicalfunctional studies (FIG. 5 a). To minimize susceptibility artefactduring MRI scanning, the implanted cannula, optical fibre andaccessories were custom-fabricated from magnetic resonance-compatiblematerials. Confocal imaging (FIG. 5 b) and optrode recording(simultaneous optical stimulation and electrical recording) under1.3-1.5% isoflurane anaesthesia’ (FIG. 5 c) were conducted to validatethe expression and functionality, respectively, of the ChR2-EYFP(enhanced yellow fluorescent protein) fusion under these conditions. Inline with previous optogenetic studies”, 473 nm light pulses at 20 Hz(15 ms pulse width) delivered through the optical fibre were found todrive local neuronal firing reliably in vivo (FIG. 5 c).

To assess fMRI signals, we acquired 0.5 mm coronal slices centered onM1, >10 days after virus injection (FIG. 5 d). Intubated animals wereplaced on a custom-designed MRI-compatible cradle with a stereotaxicframe and ventilated with 1.3-1.5% isoflurane. A 3.5 cm-diametercustom-designed transmit/receive single-loop surface coil was opposed tothe cranium and a long 300-μm diameter optical fibre inserted throughthe implanted cannula; in this configuration, the cradle with the animalwas placed into the isocentre of the magnet while the laser diode itselfwas maintained outside the 5 Gauss perimeter. To minimize systemicphysiological confounds, the ventilation volume, frequency, end-tidalCO₂ and rectal temperature levels were carefully maintained at narrowlevels known to produce most robust and reproducible BOLD signals inanaesthetized animals (3.0-3.5 cm³ per stroke, 50-60 strokes per min,3.5%, 34-38° C.). fMRI scans were performed at 7.0 Tesla (T) fieldstrength using conventional gradient-echo (GRE)-BOLD fMRI and pass-bandbalanced steady-state free precession (b-SSFP)-fMRI. Both pulsesequences were designed to have 3.5×3.5 cm² in-plane field of view(FOV), 0.5×0.5×0.5 mm³ spatial resolution and 3 s temporal resolution.GRE-BOLD fMRI was designed to be a two-dimensional, multi-slice,gradient-echo sequence with four-interleave spiral readout; 750 msrepetition time (TR) and 12 ms echo time (TE) resulting in 23 slicescovering 1.15 cm slice direction volume. This specific design allowedlarge-volume mapping of the brain during optogenetic control with hightemporal resolution.

Light pulses at 20 Hz (473 nm, 15 ms pulse width) were delivered totargeted CaMK11ix-expressing principal neurons, and in response, robustoptically-evoked BOLD signals were observed in cortical grey matter atthe virus injection and optical stimulation site, whereas in controlanimals (injected with saline instead of opsin-AAV) no detectable BOLDsignal could be elicited (FIG. 5). Stimulus-synchronized BOLDhaemodynamic responses from activated M1 voxels are displayed in FIG. 5d, and mean optogenetic fMRI haemodynamic response functions (ofMRI-HRF)in FIG. 4. Evoked BOLD was dominated by positive signals while drivingthese excitatory CaMKIIα-positive cells; in contrast, optically drivinginhibitory parvalbumin-positive cells, which may have uniqueconnectivity with local neuronal circuitry or vasculature, additionallygave rise to a zone of negative BOLD, consistent with the GABAergicphenotype, surrounding the local positive BOLD signal (See SupplementaryFIG. 4 of “Global and local fMRI signals driven by neurons definedoptogenetically by type and wiring,” Nature, Vol. 465, 10 Jun. 2010, pp,788-792). Strikingly, the BOLD dynamics observed by optically drivingthe defined CaMKIIα principal cell population embedded within the mixedM1 cell population precisely matched the dynamics of conventionalstimulus-evoked BOLD-fMRI. In particular, the ofMRI-HRF signal onsetoccurred after 3 s but within 6 s of stimulus onset; likewise offset wasreflected by a drop in BOLD signal contrast beginning within 6 s andreturning to baseline in ˜20 s after optical stimulation (FIG. 4; upperpanels: n=3, lower panels: n=8). Finally, the pronounced post-stimulusundershoot observed during systemic somatosensory stimulation in humansand animals was preserved in ofMRI-HRFs as well (FIG. 4). All of thesedynamic properties derived from driving a defined, specific (SeeSupplementary FIG. 1a of “Global and local fMRI signals driven byneurons defined optogenetically by type and wiring,” Nature, Vol. 465,10 Jun. 2010, pp, 788-792) cell population correspond closely toprevious measurements on conventional sensory-evoked BOLD.

To study macrocircuit properties of the brain using optogenetic fMRI, itwill be important to assess feasibility of monitoring long-rangeactivity in synaptically connected brain areas. MRI-compatibleelectrodes for local stimulation represent a major advance but inaddition to driving all local excitatory, inhibitory and modulatory celltypes, will also antidromically drive non-local cells that happen tohave axons within the stimulated region, posing a challenge forfunctional mapping using BOLD. We therefore used high-resolution fMRIslices capturing thalamic nuclei (coronal slices shown in FIG. 6 a) tomonitor downstream responses during optical stimulation of M1 corticalneurons. FIG. 6 b illustrates the observed specific ChR2 expression incortico-thalamic axonal projection fibers whereas thalamic cell bodiesshowed no ChR2 expression, as expected from the cortical injectionprotocol (See Supplementary FIG. 5 of “Global and local fMRI signalsdriven by neurons defined optogenetically by type and wiring,” Nature,Vol. 465, 10 Jun. 2010, pp, 788-792). Local optical stimulation was thendelivered to the cortex during fMRI, to determine if unidirectionallytriggered BOLD responses could be observed and measured (this methodeliminates the antidromic drive confound from which electricalstimulation suffers, thereby allowing true global causal connectivitymapping). FIG. 6 c and d summarize the thalamic ofMRI-HRFs; robustthalamic BOLD signals in response to MI stimulation were observed, butwith properties quite distinct from the intracortical CaMKIIα+ responsedescribed above. A markedly reduced initial rise and slope for onsetkinetics of positive-BOLD downstream thalamic recruitment was observed(FIG. 6 d, black traces; local cortical BOLD signals shown forcomparison, grey traces; cortical BOLD activation is shown in FIG. 5).

Given the unusual kinetics, we sought to determine if this delayedthalamic BOLD response would be discrepant with local thalamicelectrical activity, assessed with simultaneous optrodestimulating/recording in motor cortex and electrode recoding in thalamus(FIG. 6 e). However, a strikingly similar pattern was observed withdirect recording in thalamus, including a commensurate delay inspike-rate increase for thalamic neurons compared to cortical neuronsduring cortical optogenetic drive (FIG. 6 f), further supporting thetight correspondence between positive BOLD and local neuronalexcitation. Additional characterization showed that after this ˜5 sdelay presumably related to network properties, successfully evokedspikes recorded in the thalamus reliably followed cortical spikes byseveral milliseconds, as expected (FIG. 6 g). Summary data on mean spikerates is presented in FIG. 6 h, and on spike rate dynamics in FIG. 6 i;further details on the pass-band bSSFP-fMRI method we developed forsmall animal imaging with more robust whole-brain mapping capabilitythan traditional BOLD are presented in the Supplementary Material,particularly Supplementary FIG. 3, of “Global and local fMRI signalsdriven by neurons defined optogenetically by type and wiring,” Nature,Vol. 465, 10 Jun. 2010, pp, 788-792.

Because true functional outputs of genetically defined neurons in abrain region can be globally mapped with ofMRI (FIG. 6), it isconceivable that additional levels of specificity could also beachieved. For example, M1 excitatory pyramidal neurons form agenetically and anatomically defined class of cell, but within thisclass are cells that each project to different areas of the brain orspinal cord and therefore have fundamentally distinct roles. Genetictools may not advance far enough to separate all of these different cellclasses, pointing to the need for other promoter-independent targetingmethods. But ofMRI raises the current possibility of globally mappingthe causal roles of these cells, accessing them by means of connectiontopology—that is, by the conformation of their functional projectionpatterns in the brain. We therefore sought to test this possibility byselectively driving the M1 CaMKIIα-expressing cells that project to thethalamus.

An optical fibre was stereotactically placed in the thalamus of animalsthat had received M1 cortical viral injections (FIG. 7 a); post hocvalidation (FIG. 7 b) confirmed ChR2 expression in cortical neurons andin cortico-thalamic projection fibers. ChR2 readily triggers spikes inilluminated photosensitive axons that both drive local synaptic outputand back-propagate throughout the axon to the soma of the stimulatedcell; note that unlike the case with electrical stimulation, specificityis maintained for driving the targeted (photosensitive) axons, andtherefore this configuration in principle allows ofMRI mapping duringselective control of the M1 cortical cells that project to the thalamus.Indeed, robust BOLD signals were observed both locally in the thalamus(FIG. 7 c, coronal slices 7-12) and also in M1 (FIG. 7 d, coronal slices1-6), consistent with the anticipated recruitment of the topologicallytargeted cells both locally and distally. These data demonstrate thatChR2-expressing axonal fibre stimulation alone is sufficient to elicitBOLD responses in remote areas, and illustrate the feasibility for invivo mapping of the global impact of cells defined not only byanatomical location and genetic identity, but also by connectiontopology.

We further explored the global mapping capabilities of ofMRI. It hasbeen suggested that thalamic projections to the motor cortex may be morelikely than those to the sensory cortex, to involve both ipsilateral andcontralateral pathways, because in many cases motor control and planningmust involve bilateral coordination. This principle is challenging toassess at the functional level, because electrode-based stimulation willdrive antidromic as well as orthodromic projections, and hence maymistakenly report robust cortico-thalamic rather than thalamocorticalprojections. We therefore sought to globally map functional connectivityarising from the initial drive of anterior or posterior thalamic nucleusprojections, using ofMRI. After injecting CaMKIIα::ChR2 into thethalamus (FIG. 8), we found that optical stimulation of posteriorthalamic nuclei resulted in a strong BOLD response, both at the site ofstimulation as expected and in the posterior ipsilateral somatosensorycortex (S2) (FIG. 8 a-d). Optically stimulating excitatory cell bodiesand fibres in the more anterior thalamic nuclei resulted in BOLDresponse at the site of stimulation and also significant ipsilateral andcontralateral cortical BOLD responses (FIGS. 8 e, f), consistent withthe proposed bilaterality of anterior thalamocortical nuclei involvementin motor control and coordination Together, these results illustrate thepower of optogenetic fMRI in shedding light on the controversialidentification of positive BOLD signals with increased local neuronalexcitation, providing an empirical underpinning for fMRI BOLD. We alsofind that the properties of integrated optogenetics and BOLD-fMRI(ofMRI) allow for global mapping of the causal connectivity of definedneurons in specific brain regions, fundamentally extending thecapabilities of pharmacological or electrode-based methods (of course,contributions from additional cells and processes downstream of thedefined optically-triggered population are expected and indeed representan important aspect of this approach; it is, however, important to notethat absence of a BOLD signal does not prove the absence ofconnectivity). Finally, we demonstrate that ofMRI allows causalconnectivity mapping of cells defined not only genetically but also bycircuit topology, or the conformation of their connections in vivo.Together, the ofMRI methods and findings reported here provide tools andapproaches for further probing and defining the causal generation ofBOLD signals; these results may accelerate the search for globalcircuit-disease endophenotypes, as well as the dynamical mapping andreverse engineering of intact neural circuitry.

The skilled artisan would understand that various steps and articlesdisclosed above for such embodiments can be used selectively to effectdifferent but related embodiments.

While the present disclosure is amenable to various modifications andalternative forms, specifics thereof have been shown by way of examplein the drawings and will be described in further detail. It should beunderstood that the intention is not to limit the disclosure to theparticular embodiments and/or applications described. On the contrary,the intention is to cover all modifications, equivalents, andalternatives falling within the spirit and scope of the presentdisclosure.

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What is claimed is:
 1. A method comprising: modifying a target neuralcell population in a first region of a brain to express alight-responsive opsin polypeptide; stimulating, using a light pulse,the light-responsive opsin polypeptide in the target neural cellpopulation; scanning multiple regions of the brain via functionalmagnetic resonance imaging to observe a neural reaction in response tothe stimulation in at least one of the multiple regions of the brainand, in response, determine whether neural projections in a secondregion of the brain are connected to at least some of the modifiedtarget cell population in the first region of the brain.
 2. The methodof claim 1, further including stimulating the light-responsivepolypeptide at a first light pulse rate and a second light pulse rate.3. The method of claim 1, wherein the first region of the brain is inthe motor cortex.
 4. The method of claim 1, further includingcalibrating the stimulation of the light- responsive opsin polypeptideto provide a response in the second region within a desired range ofresponses.
 5. The method of claim 1, wherein the observed neuralreactions are used to determine a treatment plan for a disease effectingat least one of the first or second regions of the brain.
 6. The methodof claim 1, further comprising introducing a drug into the brain, andrepeating the steps of stimulating the light-responsive opsinpolypeptide, and scanning multiple regions of the brain; and determiningthe effectiveness of the drug based on a comparison of the observedneural reactions in the scan before the introduction of the drug and theobserved neural reactions in the scan after the introduction of thedrug.
 7. The method of claim 1, further including: modifying a secondtarget neural population in the first region of the brain to express asecond type of light-responsive opsin polypeptide; stimulating thesecond type of light-responsive opsin polypeptide in the second targetneural population; scanning multiple regions of the brain via functionalmagnetic resonance imaging to observe a neural reaction in response tothe stimulation of the second neural population in at least one of themultiple regions of the brain and determine therefrom whether neuralpaths in the second region of the brain are neurally connected to atleast some of the modified second target neural populations in the firstregion of the brain.
 8. The method of claim 2, wherein the results ofthe observation of first stimulation and the second stimulation arecombined, and providing a functional map of the brain including at leastthe results of the first observation and the second observation.
 9. Amethod comprising: modifying a target neural cell population to expressa light-responsive opsin polypeptide in a first region of a brain,wherein the light-responsive opsin polypeptide modulates an activity ofthe target cell population in response to light; stimulating thelight-responsive opsin polypeptide in the target neural cell populationusing light pulses; scanning at least the first region of the brain viafunctional magnetic resonance imaging (fMRI); stimulating the targetneural cell population using an electric pulse; performing fMRI scansand observing a blood oxygenation level-dependent (BOLD) signal responseto excitation of the target cell population using electronicstimulation; and based at least in part on the BOLD signal responses inthe target cell population due to light stimulation and electronicstimulation, assessing the BOLD fMRI scan.
 10. The method of claim 1,wherein the light-responsive opsin polypeptide is a channelrhodopsin.11. The method of claim 10, wherein the light-responsive opsinpolypeptide is a ChR2 or a VChR1 channelrhodopsin.
 12. The method ofclaim 1, wherein the light-responsive opsin polypeptide is an NpHR ionpump.
 13. The method of claim 1, wherein the light-responsive opsinpolypeptide is encoded by a nucleotide sequence that is operably linkedto a neuron-specific promoter.
 14. The method of claim 13, wherein theneuron-specific promoter is a CaMKIIα promoter.
 15. The method of claim1, wherein the first region of the brain is in the thalamus.
 16. Themethod of claim 1, wherein the light is delivered by an optical fiber.17. The method of claim 1, wherein target neural cell population ismodified using a recombinant expression vector encoding thelight-responsive opsin polypeptide.
 18. The method of claim 17, whereinthe recombinant expression vector is delivered to the target neural cellpopulation via a cannula.